UC Riverside

Nuclear magnetic resonance (NMR) has proven itself to be a powerful tool for analytic, kinetic, structural, and mechanistic studies of biomolecules due to its atomic-level resolution of the local chemical environment. These studies often employ strategies related to observing chemical shifts that detail site-specific information regarding protonation states, pKa, structure, and more. The biomolecule this study is directed toward is Toho-1 β-Lactamase, a Class A extended-spectrum β-lactamase that possesses a high affinity for a wider range of antibiotics than other enzymes in its family. These enzymes are responsible for the rise in antibiotic resistance that threatens the effectiveness of long-standing β-lactam antibiotics and inhibitors by hydrolyzing the medicinally relevant β-lactam ring, effectively destroying their antibiotic properties.Novel inhibitors that do not contain a β-lactam ring, such as Avibactam, are beginning to be studied, produced, and prescribed. Although studies reliably demonstrate their safety and effectiveness, there is still uncertainty regarding exactly how these novel inhibitors function. To better understand these inhibitors and aid in the production of more novel, effective ones, this work predominantly employs NMR Crystallography to investigate the avibactam-inhibited active site of Toho-1 β-Lactamase.NMR Crystallography integrates solid-state NMR, x-ray crystallography, and first-principles calculations to provide testable models that accurately reflect the chemical environment of enzyme active sites by comparing theoretical chemical shifts derived from structural models with experimental chemical shifts. This work is also facilitated by our nearly complete backbone and side-chain assignment, enabled by state-of-the-art, high-resolution, 13C-detected solid-state NMR spectra. Utilizing these techniques, we aim to describe, down to the protonation states of key amino acids, the active site of avibactam-bound Toho-1 β-Lactamase to better understand avibactam-led inhibition.This work also highlights recent advancements in solid-state NMR by showcasing the capabilities brought forward by MAS-Cryoprobes. The sensitivity enhancement provides opportunities to detect weak and broad signals that remain undetected even at magnet strengths near the commercially available limit. We most specifically use this development to investigate lysine side chains of Toho-1 β-Lactamase.